The invention resides in the technical fields of immunology and medicine.
Alzheimer""s disease (AD) is a progressive disease resulting in senile dementia. See generally Selkoe, TINS 16,403-409 (1993); Hardy et al., WO 92/13069; Selkoe, J. Neuropathol. Exp. Neurol. 53, 438-447 (1994); Duff et al., Nature 373, 476-477 (1995); Games et al., Nature 373, 523 (1995). Broadly speaking, the disease falls into two categories: late onset, which occurs in old age (65+ years) and early onset, which develops well before the senile period, i.e., between 35 and 60 years. In both types of disease, the pathology is the same but the abnormalities tend to be more severe and widespread in cases beginning at an earlier age. The disease is characterized by at least two types of lesions in the brain, senile plaques and neurofibrillary tangles. Senile plaques are areas of disorganized neuropil up to 150 xcexcm across with extracellular amyloid deposits at the center visible by microscopic analysis of sections of brain tissue. Neurofibrillary tangles are intracellular deposits of microtubule associated tau protein consisting of two filaments twisted about each other in pairs.
The principal constituent of the plaques is a peptide termed Axcex2 or xcex2-amyloid peptide. Axcex2 peptide is an internal fragment of 39-43 amino acids of a precursor protein termed amyloid precursor protein (APP). Several mutations within the APP protein have been correlated with the presence of Akheimer""s disease. See, e.g., Goate et al., Nature 349, 704) (1991) (valine717 to isoleucine); Chartier Harlan et al. Nature 353, 844 (1991)) (valine717 to glycine); Murrell et al., Science 254, 97 (1991) (valine717 to phenylalanine); Mullan et al., Nature Genet. 1, 345 (1992) (a double mutation changing lysine595-methionine596 to asparagine595-leucine596). Such mutations are thought to cause Alzheimer""s disease by increased or altered processing of APP to Axcex2, particularly processing of APP to increased amounts of the long form of Axcex2 (i.e., Axcex21-42 and Axcex21-43). Mutations in other genes, such as the presenilin genes,PS1 and PS2, are thought indirectly to affect processing of APP to generate increased amounts of long form Axcex2 (see Hardy, TINS 20, 154 (1997)). These observations indicate that Axcex2, and particularly its long form, is a causative element in Alzheimer""s disease.
McMichael, EP 526,511, proposes administration of homeopathic dosages (less than or equal to 10xe2x88x922 mg/day) of Axcex2 to patients with preestablished AD. In a typical human with about 5 liters of plasma, even the upper limit of this dosage would be expected to generate a concentration of no more than 2 pg/ml. The normal concentration of Axcex2 in human plasma is typically in the range of 50-200 pg/ml (Seubert et al., Nature 359, 325-327 (1992)). Because EP 526,511""s proposed dosage would barely alter the level of endogenous circulating Axcex2 and because EP 526,511 does not recommend use of an adjuvant, as an immunostimulant, it seems implausible that any therapeutic benefit would result.
By contrast, the present invention is directed inter alia to treatment of Alzheimer""s and other amyloidogenic diseases by administration of fragments of Axcex2, or antibody to certain epitopes within Axcex2 to a patient under conditions that generate a beneficial immune response in the patient. The invention thus fulfills a longstanding need for therapeutic regimes for preventing or ameliorating the neuropathology and, in some patients, the cognitive impairment associated with Alzheimer""s disease.
This application is related to International Application No. PCTUS00/14810 filed May 26, 2000. Publication No. WO 00/72880: U.S. application Ser. No. 09/322,289, filed May 28, 1999; PCT/US98/25386, filed Nov. 30, 1998, Publication No. WO 99/27944; U.S. applilication Ser. No. 09/201,430, filed Nov. 30, 1998; U.S. application Ser. No. 60/067,740, filed Dec. 2, 1997; and, U.S. application Ser. No., 60/080,970 filed Apr. 7, 1998; each of which is incorporated by reference in its entirety for all purposes.
In one aspect, the invention provides methods of preventing or treaang a disease associated with amyloid deposits of Axcex2 in the brain of a patient. Such diseases include Alzheimer""s disease, Down""s syndrome and cognitive impairment. The latter can occur with or without other characteristics of an amyloidogenic disease. Some methods of the invention entail administering an effective dosage of an antibody that specifically binds to a component of an amyloid deposit to the patient. Such methods are particularly useful for preventing or treating Alzheimer""s disease in human patients. Some methods entail administering an effective dosage of an antibody that binds to Axcex2. Some methods entail administering an effective dosage of an antibody that specifically binds to an epitope within residues 1-10 of Axcex2. In some methods, the antibody specifically binds to an epitope within residues 1-6 of Axcex2. In some methods, the antibody specifically binds to an epitope within residues 1-5 of Axcex2. In some methods, the antibody specifically binds to an epitope within residues 1-7 of Axcex2. In some methods, the antibody specifically binds to an epitope within residues 3-7 of Axcex2. In some methods, the antibody specifically binds to an epitope within residues 1-3 of Axcex2. In some methods, the antibody specifically binds to an epitope within residues 1-4 of Axcex2. In some methods, the antibody binds to an epitope comprising a free N-terminal residue of Axcex2. In some methods, the antibody binds to an epitope within residues of 1-10 of Axcex2 wherein residue 1 and/or residue 7 of Axcex2 is aspartic acid. In some methods, the antibody specifically binds to Axcex2 peptide without binding to full-length amyloid precursor protein (APP). In some methods, the isotype of the antibody is human IgG1.
In some methods, the antibody binds to an amyloid deposit in the patient and induces a clearing response against the amyloid deposit. For example, such a clearing response can be effected by Fc receptor mediated phagocytosis.
The methods can be used on both asymptomatic patients and those currently showing symptoms of disease. The antibody used in such methods can be a human, humanized, chimeric or nonhuman antibody and can be monoclonal or polyclonal. In some methods, the antibody is prepared from a human immunized with Axcex2 peptide, which human can be the patient to be treated with antibody.
In some methods, the antibody is administered with a pharmaceutical carrier as a pharmaceutical composition. In some methods, antibody is administered at a dosage of 0.0001 to 100 mg/kg, preferably, at least 1 mg/kg body weight antibody. In some methods, the antibody is administered in multiple dosages over a prolonged period, for example, of at least six months. In some methods, the antibody is administered as a sustained release composition. The antibody can be administered, for example, intraperitoneally, orally, subcutaneously, intcacranially, intramuscularly, topically, intranasally or intravenously.
In some methods, the antibody is administered by administering a polynucleotide encoding at least one antibody chain to the patient. The polynucleotide is expressed to produce the antibody chain in the patient. Optionally, the polynucleotide encodes heavy and light chains of the antibody. The polynucleotide is expressed to produce the heavy and light chains in the patient. In some methods, the patient is monitored for level of administered antibody in the blood of the patient.
In another aspect, the invention provides methods of preventing or treating a disease associated with amyloid deposits of Axcex2 in the brain of patient For example, the methods can be used to treat Alzheimer""s disease or Down""s syndrome or cognitive impairment. Such methods entail administering fragments of Axcex2 or analogs thereof eliciting an immunogenic response against certain epitopes within Axcex2. Some methods entail administering to a patient an effective dosage of a polypeptide comprising an N-terminal segment of at least residues 1-5 of Axcex2, the first residue of Axcex2 being the N-terminal residue of the polypeptide, wherein the polypeptide is free of a C-terminal segment of Axcex2. Some methods entail administering to a patient an effective dosage of a polypeptide comprising an N-terminal segment of Axcex2, the segment beginning at residue 1-3 of Axcex2 and ending at residues 7-11 of Axcex2. Some methods entail administering to a patient an effective dosage of an agent that induces an immunogenic response against an N-terminal segment of Axcex2, the segment beginning at residue 1-3 of Axcex2 and ending at residues 7-11 of Axcex2 without inducing an immunogenic response against an epitope within residues 12-43 of Axcex243.
In some of the above methods, the N-terminal segment of Axcex2 is linked at its C-terminus to a heterologous polypeptide. In some of the above methods, the N-terminal segment of Axcex2 is linked at its N-terminus to a heterologous polypeptide. In some of the above methods, the N-terminal segment of Axcex2 is linked at its N and C termini to first and second heterologous polypeptides. In some of the above methods, the N-terminal segment of Axcex2 is linked at its N terminus to a heterologous polypeptide, and at its C-terminus to at least one additional copy of the N-terminal segment. In some of the above methods, the heterologous polypeptide and thereby a B-cell response against the N-terminal segment. In some of the above methods, the polypeptide further comprises at least one additional copy of the N-terminal segment. In some of the above methods, the polypeptide comprises from N-terminus to C-terminus, the N-terminal segment of Axcex2, a plurality of additional copies of the N-terminal segment, and the heterologous amino acid segment. In some of the above methods, the N-terminal segment consists of Axcex21-7. In some of the above methods, the N-terminal segment consists of Axcex23-7.
In some methods, the fragment is free of at least the 5 C-terminal amino acids in Axcex243. In some methods, the fragment comprises up to 10 contiguous amino acids from Axcex2. Fragments are typically administered at greater than 10 micrograms per dose per patient.
In some methods, the fragment is administered with an adjuvant that enhances the immune response to the Axcex2 peptide. The adjuvant and fragment can be administered in either order or together as a composition. The adjuvant can be, for example, aluminum hydroxide, aluminum phosphate, MPL(trademark), QS-21 (Stimulon(trademark)) or incomplete Freund""s adjuvant
The invention further provides pharmaceutical compositions comprising fragments of Axcex2 or other agents eliciting immunogenic response to the same epitopes of Axcex2, such as described above, and an adjuvant. The invention also provides pharmaceutical compositions comprising any of the antibodies described above and a pharmaceutically acceptable carrier.
In another aspect, the invention provides methods of screening an antibody for activity in treating a disease associated with deposits of Axcex2 in the brain of a patient (e.g., Alzheimer""s disease). Such methods entail contacting the antibody with a polypeptide comprising at least five contiguous amino acids of an N-terminal segment of Axcex2 beginning at a residue between 1 and 3 of Axcex2, the polypeptide being free of a C-terminal segment of Axcex2. One then determines whether the antibody specifically binds to the polypeptide, specific binding providing an indication that the antibody has activity in treating the disease.
In another aspect, the invention provides methods of screening an antibody for activity in clearing an antigen-associated biological entity. Such methods entail combining the antigen-associated biological entity and the antibody and phagocytic cells bearing Fc receptors in a medium. The amount of the antigen-associated biological entity remaining in the medium is then monitored. A reduction in the amount of the antigen-associated biological entity indicates the antibody has clearing activity against the antigen-associated biological entity. The antigen can be provided as a tissue sample or in isolated form. For example, the antigen can be provided as a tissue sample from the brain of an Alzheimer""s disease patient or a mammal animal having Alzheimer""s pathology. Other tissue samples against which antibodies can be tested for clearing activity include cancerous tissue samples, virally infected tissue samples, tissue samples comprising inflammatory cells, nonmalignant abnormal cell growths, or tissue samples comprising an abnormal extracellular matrix.
In another aspect, the invention provides methods of detecting an amyloid deposit in a patient. Such methods entail administering to the patient an antibody that specifically binds to an epitope within amino acids 1-10 of Axcex2, and detecting presence of the antibody in the brain of the patient In some methods, the antibody binds to an epitope within residues 4-10 of Axcex2. In some methods, the antibody is labelled with a paramagnetic label and detected by nuclear magnetic resonance tomography.
The invention further provides diagnostic kits suitable for use in the above methods. Such a kit comprises an antibody that specifically binds to an epitope with residues 1-10 of Axcex2. Some kits bear a label describing use of the antibody for in vivo diagnosis or monitoring of Alzheimer""s disease.